J.Med. Chem. 1995,38,803-809
803
[[[(Thienylcarbonyl)alkylloxylphenyll-and [[[(Pyrrylcarbonyl)alkyl]oxy]phenyl]oxazoline Derivatives with Potent
and
Selective Antihuman Rhinovirus Activity1 Silvio Massa,ll Federico Corelli,t Marino Artico,*JlAntonello Mai,” Rino Ragno,* Antonella De Montis,? Anna Giulia Loi,t Simona Corrias,t Maria Elena Marongiu,? and Paolo La Collat Dipartimento d i Studi Farmaceutici, Universith di Roma “La Sapienza”, P.le A. Mor0 5, 00185 Roma, Italy, Dipartimento Farmaco Chimico Tecnologico, Universith d i Siena, Banchi d i Sotto 55, 53100 Siena, Italy, Dipartimento d i Studi d i Chimica e Tecnologie delle Sostanze Biologicamente Attive, P.le A. Mor0 5, 00185 Roma, Italy, and Dipartimento di Biologia Sperimentale, Sezione d i Microbiologia, Universith d i Cagliari, v.le Regina Margherita 45, 09124 Cagliari, Italy Received December 28, 1993@
As a n approach to more extensive structural modifications of [(oxazolylphenoxy)alkyllisoxazoles, we synthesized new compounds characterized by the replacement of the isoxazole nucleus with furan, pyrrole, and thiophene rings and by the presence of a ketocarbonyl group in the aliphatic chain connecting these pentatomic heterocycles to the 4-(4,5-dihydro-2-oxazolyl)phenoxy, 4-(ethoxycarbonyl)phenoxy,and 4-carboxyphenoxy moieties. Some pentamethylene derivatives were also prepared, and their antirhinovirus activity was compared to that of the corresponding ketomethylene derivatives. Syntheses were carried out by Friedel-CraRs acylation of the above pentatomic heterocycles and subsequent reaction of chloroalkyl ketones with the proper 4-substituted phenol. Reduction of the ketone function afforded the related polymethylene derivatives. The new compounds were tested for antirhinovirus activity and cytotoxicity in comparison with WIN 51711, used as reference drug. Inspection of the structure-activity relationships revealed t h a t the thiophene ring and the carbonyl group are the structural components which to a large extent contribute to the positive biological profile in terms of both wideness of spectrum and low cytotoxicity. Among the various derivatives, compounds 8e,d showed in vitro the same potency of WIN 51711 but a cytotoxicity at least 10 times lower. Rhinoviruses (HRVs) are the most frequent etiological agents of common cold and mild localized infections of the upper respiratory tract in humans (about 40% of cases). Considering their worldwide diffusion, these syndromes have relevant socioeconomic importance and many efforts have been directed toward the identification of agents useful in the prophylaxis and therapy of HRV infections. Nevertheless, the inherent heterogeneity of this group of human pathogens, demonstrated by the isolation so far of over 100 different HRV serotypes,a has hampered the development of vaccines and made troublesome the design of effective, broad-spectrum drugs. A valuable contribution to the synthesis of antirhinovirus agents has been provided by Diana and cow o r k e r ~ . ~Chemical -~ elaboration of the prototypical structure of arildone has led to the development of [(oxazolylphenoxy)alkyl]isoxazoles 1, a class of potent, broad-spectrum antipicornavirus agents, which differ in the length of the aliphatic chain connecting the oxazolylphenoxy and isoxazole moieties and present a variety of substituents at different positions in the phenyl and/or oxazoline rings. Disoxaril(1, n = 7, X = R = H), in particular, has been found active in vitro against several entero and rhinovirus serotypes. X-ray crystallographic studies of drug-human rhinovirion complexes have shown that [(oxazolylphenoxy)alky11Dpt Studi Farmaceutici. Dpt Farmaco Chimico Tecnologico. t Dpt Studi Chimica Tecnologie Sostanze Biologicamente Attive. + Dpt Biologia Sperimentale, Sezione Microbiologia. @Abstractpublished in Advance ACS Abstracts, January 1, 1995.
11
0
isoxazoles bind in a hydrophobic pocket beneath the canyon floor and either block the uncoating process by stabilizing the virion structure or lead to conformational changes which affect the putative viral receptor binding site, thus preventing virion adsorption to cell receptors. 9-1 H3C I C H Z ~ - O & -~ ~
R
1
Although extensive structure-activity studies have been carried out on this class of antipicornavirus agents by Diana and co-workers, t o the best of our knowledge no attempt has been made to ascertain whether the isoxazole nucleus really assures the best interaction of the above compounds with the specific amino acids of the viral capsid protein 1 (VP1) hydrophobic pocket. This aspect seemed to us to be of prominent interest, since each of the rhinovirus serotypes differs to varying degree in the VP1 amino acid sequence. Accordingly, as an approach t o more extensive structural modifications of [(oxazolylphenoxy)alkyl]isoxazoles, we synthesized derivatives 2 characterized by the replacement of the isoxazole nucleus with pyrrole, thiophene, and furan rings and by the presence of a ketocarbonyl group in the aliphatic chain connecting these pentatomic hetero44ethoxcycles to the 4-(4,5-dihydro-2-oxazolyl)phenoxy, ycarbonyl)phenoxy,or 4-carboxyphenoxy moieties. Some pentamethylene analogues having the formula 3 were also prepared and tested for a comparison with the
0022-262319511838-0803$09.00/0 0 1995 American Chemical Society
804 Journal of Medicinal Chemistry, 1995,Vol. 38,No.5
Scheme la
Massa et al.
Scheme 2a
d R
/qv 1 1 d ,e R' = 2-oxarolinyl 12d,e R'=COOC2H5
N
mchodA
mclhod B
I S02Ph
R
4g
1 5 R = SO2Ph 16 R = H
bL
N
H
17 R
=
A !,
N 18 R = C O O C 2 H 5
method C
1Ud,e
x 4a,b,c'.f'
L mehod A
Y=H2
7a,b,d-g R'=2-oxazolinyl 8 C - I : R'=COOCzHs 9 R'= CONHCH2CHzOH; R = 5-CH3: X = 0 a Compounds: a,X = NH,R = H; b,X = NCH3, R = H; c, X = 0, R = 5-CH3; c', X = 0, R = 2-CH3; d, X = S, R = 541; d', X = S, R = 241; e, X = S, R = 5-CH3; e', X = S, R = 2-CHs; f, X = S, R = 3-CH3; f,X = S, R = 3-CH3; g, X = S, R = 4-CH3. Reagents: (a) Cl(CHz)&OCl, del$ (b) 6a, KzCO3, NaI, CH3CN; (c) 6b, KzC03, NaI, CH&N; (d) 6 q KzCO3, NaI, CH3CN (e) LiAlb, AlC4; (0 KOH, CzH50H.
H3Ce
R
2 Y=O
3
Y = H ~
n=3,4 m=5 X = 0. NH. N C H 3 , S Z = COOH
COOC~HS
T3 In a preliminary communication12 we have reported on the potent and selective anti-HRV-14activity of some compounds of formula 2. When tested for antiviral activity, some thiophene, pyrrole, and furan derivatives were found inactive against Coxsackie B1 and marginally active against polio but showed potent and selective activity against HRV-14. In the present paper we give full account on the synthesis of a larger group of nonisoxazole analogues 2 and 3, as well as on their activity against a wider spectrum of HRV serotypes, and highlight some aspects of their structure-activity relationships.
Chemistry Friedel-Crafts acylation (Scheme 1)of the electronrich heteroaromatics 4a-f with 5-chlorovaleryl chloride afforded the heteroaryl ketones 5a-g, which readily underwent nucleophilic displacement by 4-(4,5-dihydro2-oxazolyl)phenol (6a),6ethyl 4-hydroxybenzoate (6b), and N-(4-hydroxybenzoyl)ethanolamine ( 6 ~ in ) ~the presence of potassium carbonate and sodium iodide to give compounds 7a,b,d-g, 8c-g, and 9, respectively. Similarly, reaction of 6a,b with the haloalkyl derivatives
H
20a R = H 20b R = C H 3
method d D
3
H3C
R
I
R = H. CI. C H 7 , C H O R'=H,CH3
,
C
C
,
H
3
R
P
I
I
24
RYA7
25 26
R=COOC2Hs R=COOH
CH3
4b
a
21a R = H 21b R = C H 3
22a R = H R = C O O C ~ H S 2 2 b R = H: R = 2-oxazolinyl 2 2 c R = CH3: R = COOC2H5 2 2 d R = CH3: R' = 2. oxazolinyl
corresponding ketomethylene analogues 2. Y
1Ya R = H 1Yb R = C H 3
4h R = H 4i R=CH3
2CH3 3 methodgc
G
(a) Cl(CHz)&OCl, Mc13; (b) NaOH, HzO, dioxane; (c) 6a, KzC03, NaI, CHsCN; (d) 6a or Bb,DEAD,PbP (e) (COC112, DMF, then Cl(CHz)&OCl, AlCl3; (f) Bb,KzCO3, NaI, CH3CN; (g) KOH, CzH50H.
10d,e, in turn obtained by reduction of 5d,e with lithium aluminum hydridelaluminum trichloride, provided compounds lld,e and 12d,e, respectively. Finally, esters 8d-g and 12d were hydrolyzed to the corresponding carboxylic acids 13d-g and 14. The synthesis of the compounds bearing the 3-pyrrolyl moiety as the heterocyclic head was performed following the procedures outlined in Scheme 2. 1-(Phenylsulfony1)-W-pyrrole (4g)13was acylated with 5-chlorovaleryl chloride, essentially as reported by Kakushima and cow o r k e r ~for~ similar ~ cases, to give the intermediate 15, which on treatment with sodium hydroxide in 50% aqueous 1,Cdioxane lost the benzenesulfonyl group to provide 3-(5-chloropentanoyl)-~-pyrrole (16). Subsequent reaction with 6a or 6b led to compounds 17 and 18, respectively. When the intermediates 19a,b, obtained by acylation of 2,5-dimethyl-W-pyrrole (4h)and 1,2,5-trimethyl-Wpyrrole (49, were reacted with 6a or 6b, only minor amounts of the expected compounds 22a-d were isolated, the main reaction products being derivatives 21a,b. These are likely to arise during the aqueous workup of the reaction from the hydrolysis of dienol
Thienyl and Pyrryl Compounds with Antirhinovirus Activity
Scheme 3" Br(CHZ)SBr
* 0-Br
b
method E
method E n
r c l
r
1
method D
33
(a) Cl(CH2)3COCl, Mc13; (b) KzCO3, NaI, CH3CN; (c) NaH, anhydrous dioxane; (d) 6b or 6a,DEAD, Ph3P. a
ether intermediates 20a,b, whose formation can be explained by a base-mediated abstraction of a proton from the 2-methyl group of 19a,bto generate a dienolate anion which undergoes intramolecular 0-alkylation. Hence the preparation of compounds 22a-d was performed under neutral conditions by means of a Mitsunobu reaction between 21a,b and the suitably substituted phenols 6a,b. When 1-methyl-1H-pyrrole (4b) was treated successively with the Vilsmeier-Haack reagent, generated from N,N-dimethylformamidefphosphorylchloride, and 5-chlorovaleryl chloride/aluminum trichloride complex,15J6 after treatment with water, 4-(5-chloropentanoyl)-l-methyl-1H-pyrrole-2-carboxaldehyde (23)was obtained, which was reacted with 6a,b to afford derivatives 24 and 25, respectively. Alkaline hydrolysis of the latter compound led to the carboxylic acid 26. Alkylation of 6a with an excess of 1,5-dibromopentane (Scheme 3) gave the intermediate 27, which, upon treatment with pyrrole or pyrrolidine in the presence of sodium hydride, furnished compounds 28 and 29, respectively. Finally, acylation of 2-methylthiophene (4e') with 4-chlorobutanoyl chloride followed by reaction of the intermediate 30 with 6a,b gave only minor amounts of compounds 32 and 33. These latter were obtained in moderate to good yield from the intermediate 31 by the Mitsunobu reaction with 6a,b, respectively, analogously to that described above for derivatives 19a,b. Chemical and physical data of the new compounds are reported in Table 1.
Results and Discussion Compounds were screened for anti-HRV activity in a tetrazolium-based colorimetric assay (MTT assay) using
Journal of Medicinal Chemistry, 1995, Vol. 38,No.5 805
HeLa-Ohio cells. In the antiviral assays we used the same 15 serotypes that had been chosen to test the activity of [(oxazolylphenoxy)alkyllisoxazoles. Whenever the compounds showed a broad spectrum of antiHRV activity, MIC~OS (i.e., compound doses that inhibit 80% of the serotypes tested) were the basis for S A R considerations. In the other cases, EC50 values were used. Tables 2-4 show the cytotoxicity and anti-HRV activity of the new compounds together with those obtained for WIN 51711, used as reference drug. Among the furan derivatives (Table 21, compound 8c showed activity against HRV-2 and HRV-14 (EC50s = 0.9 and 2.4 pM, respectively) comparable to those of WIN 51711 (EC~OS = 2.3 and 1.7 pM, respectively). Unfortunately, the limited amount of this drug made it impossible to extend the assays to additional serotypes. Compounds 18 and 25 were the only pyrrole derivatives with a wide spectrum of anti-HRV activity; the latter was more potent with an MICso of 8.4 pM (2.4fold higher than that of WIN 51711). Although differently substituted, both pyrrole rings were linked to the ketomethylene chain a t the 3 position and both compounds were carbethoxy derivatives. Interestingly, when the carbethoxy group was substituted by the oxazoline (see compounds 17 and 24), a loss of antiviral activity was observed. A n inactive derivative was also obtained by replacing the carbethoxy group with a carboxyl group (compare compounds 25 and 26). In the thiophene series, compound 8e (Table 3) and 8d (Table 4) were the most potent against a wide spectrum of HRV serotypes, with MICBOS of 2.3 and 2.1 pM, respectively, which were slightly lower than that of WIN 51711 (3 pM). Structure-activity relationships suggest that, as in the case of several WIN compounds, the anti-HRV activity of the thiophene derivatives is increased or lowered by the substitution of the oxazoline ring with a carbethoxy or carboxyl group, respectively (compare compounds 7d, 8d, and 13d; 7e, 8e, and 13e; 7f, 8f, and 13f; 7g, 8g, and 13g). Moreover, the potency of their anti-HRV activity depends on the position of the methyl substituent. In fact, in both the (oxazolylphenoxy)- (7e-g) and (carbethoxyphenoxy)alkyl (8e-g) derivatives, the anti-HRV activity progressively increases as the methyl group on the thiophene ring shifts from position 3 to position 5. However, the substitution of the 5-methyl for a 5-chloro in the thiophene ring leads to a loss of wide-spectrum anti-HRV activity in the (oxazoly1phenoxy)alkyl derivatives (compare MICsos of compounds 7d,e) but not in the (carbethoxyphenoxy)alkyl derivatives (compare MICsos of compounds 8d,e). When the MICso of 7e is compared to that of 32 and that of 8e is compared to the MICso of 33, it can be concluded that thiophene derivatives with a five-carbon chain are more potent than their counterparts with a four-carbon chain. Chlorothiophene derivatives bearing either the ethoxycarbonyl group or the oxazoline moiety were found to be equipotent. Conversely, the corresponding carboxyl derivatives always were inactive. Finally, the substitution of the carbonyl group with a further methylene group led in the thiophene series to a decrease of antiviral activity, in terms of EC50 andor MICsO,and to enhanced cytotoxicity (compare compounds 7d,e with lld,e and 8d,e with 12d,e).
806 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 5
Massa et al.
Table 1. Chemical and Physical Data of the New Compounds
R
R'
O 1 5 , 1 6 , 19, 2 1
compd Sa
X NH NCH3 0 S S S S NH NCH3 S S S
R H H 5-CH3 5-C1 5-CH3 3-CH3 4-CH3 H H 541 5-CH3 3-CH3 4-CH3 5-CH3 5-C1 5-CH3 4-CH3 3-CH3 5-CH3 5-C1 5-CH3 5-C1 5-CH3 5-C1 5-CH3 5-C1 5-CH3 4-CH3 3-CH3 5-C1 H H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
Y
R
0 1 1 , 18, 22, 24-26
Y
mp, "C recryst solventa yield, % 59-61 A 41 5b oil 50 oil 5c 45 5d 37-38 B 95 45-47 5e C 80 32-33 42 5f A oil 8 5g 155-156 7a 2-oxazolinyl D 70 2-oxazolinyl 133-134 D 7b 69 2-oxazolinyl 143-144 E 97 7d 2-oxazolinyl E 7e 129-130 25 2-oxazolinyl 108-109 C 7f 13 2-oxazolinyl 118-119 S 16 E 7g COOEt 8c 0 C 66-67 30 S COOEt 87-89 76 8d C COOEt 94-95 71 C 8e S COOEt 67-69 38 C 8f S 0 COOEt E S 0 56-57 100 8g CONHCHzCH2OH 125-127 44 9 0 0 F oil 10d S 89 H2 oil 10e S 44 H2 lld S 99-100 52 2-oxazolinyl C H2 2-oxazolinyl D lle S 96-98 59 Hz 12d S oil 71 COOEt H2 COOEt 12e S 36-38 52 G H2 169-171 COOH 13d S 0 H 66 13e S 0 187-189 93 COOH H 153-155 COOH 13f S 0 I 86 155-157 S 0 75 COOH H 13g 14 160-161 COOH H S 72 Hz c1 16 SOzPh 70-72 C 81 H 101-103 c1 J 85 16 157-159 2-oxazolinyl H 17 H D 45 H 105-107 COOEt 18 H E 88 c1 H 19a K 93-95 55 oil c1 19b 42 CH3 H 70-71 OH 21a C 39 OH 21b I 72-73 54 CH3 H 117-119 COOEt 22a E 78 (7Ib 2-oxazolinyl H 22b 118-120 E 84 (6)b 22c 63-65 COOEt E 85 (13Ib CH3 106-109 2-oxazolinyl 22d E 92 (9)b CH3 23 43-46 80 G 173-174 24 CHO 2-oxazolinyl H 42 CH3 25 CHO 117-118 COOEt H 78 CH3 181-183 26 CHO COOH L 75 CH3 70-72 27 70 A 28 105-107 A 53 29 A 69-71 72 30 oil 82 31 oil 21 32 144-145 93 (12)b D 33 85-86 C 30 (8)b a A = n-hexane. B = ethyl ether. C = cyclohexane. D = acetonitrile. E = benzene-cyclohexane. F = ethyl acetate. G = petroleum ether. H = ethanol. I = benzene. J = dichloromethane. K = carbon tetrachloride. L = toluene. Data without parentheses refer to method D; data within parentheses refer to method B. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
In summary, considering the general formula 2 of the new anti-HRV agents here described, the structural features which ensure the best biological profile seem to be (i) a thiophene ring as heterocyclic terminus, (ii) a chlorine atom or a methyl group in the a position of thiophene, (iii) a carbonyl group conjugated with
formula
CsHiaClNO
thiophene, (iv) a tetramethylene chain connecting the carbonyl group to the phenoxy moiety, and (v) an ethoxycarbonyl substituent on the benzene ring. Compounds 8d,e, which best meet these structural requirements, are the most potent among the newly synthesized derivatives against a wide range of HRV
Thienyl and Pyrryl Compounds with Antirhinovirus Activity Table 2. In Vitro Antirhinoviral Activity of Furan and Pyrrole Derivatives Compd
EGO
Svuc1ure
HRV-2
HRV-14
3.20
8.01
MlCto
CCio
>32.05
>320
Journal of Medicinal Chemistry, 1995, Vol. 38, No. 5 807 Table 3. In Vitro Antirhinoviral Activity of Methylthiophene Derivatives svucture
Compd
EC;O HRV-2 HRV-14
MK&
Oh\
&
Me-
0.91
2.42
ND
1.31
0.14
9.04
146
2.91
0.87
14.58
73
29.15
1.46
>29.15
291
0.06
0.29
2.31
>289
2.89
5.78
ND
>289
6.94
>28.90
20.80
202
2.58
11.50
>30.39
>303
1.59
7.16
14.33
301
>31.44
>31.44
>31.44
~314
>31.44
>31.44
>31.44
>314
32
15.15
22.73
>30.30
NLI
33
10.60
>30.12
>30.12
>301
2.35
1.76
3.51
37
7 e Me&
0 2
'Ir Me+
0-
65
H0-m
Me+
wEt
J y o
2 2 c Me&
>29.41
>29.41
205
2.28
16.81
>28.01
56
+fl?
>28.25
Me
e
r
Ife
e
7
M
& O,E~
flo OFH
Me
I
Me
22d
>29.41
'le M
p
11.30
>28.25
141
>28.25
>28.25
>282
Me
0
>28.25
24
N I
oHcfl Me
25
0
0.67
1.96
8.40
56
>30.90
>303
i"
o"cye-&p G"-
>30.90
26
N I
~30.90
WIN 5171 I
M
Me
28
29
1.43
>31.85
15.92
159
Table 4. In Vitro Antirhinoviral Activity of 5-Chlorothiophene Derivatives
>33.11
>33.11
>33.11
165
Compd
2.35
1.76
3.51
37
EoC: HRV-2 HRV-14
Suuc1we
MlCto
CCio
01
WIN51711
EC50: compound concentration @M) required to achieve 50% protection of HeLa cells from HRV-induced cytopathic effect. MICOO:compound concentration GM) which inhibits 80% of the serotypes tested. The serotypes used were HRV-lA, -1B, -2, -6, -14, -15, -21, -22, -25, -30, -41, -50, -67, -86, and -89. cc50: compound concentration @M) required to reduce the viability of mock-infected HeLa cells by 50%.
serotypes, showing MICso values of 2.2 and 2.3 pM, respectively, which compare well with that of WIN 51711 (3.5pM). It is worth pointing out that contrary to WIN 51711, which is cytotoxic at a concentration of 36.7 pM, most of the new compounds are far less toxic and in some cases exhibit more than a 10-fold more favorable selectivity index (SI, defined as the ratio between CCSOand MICso).
Experimental Section Chemistry. Melting points (Buchi 530 melting point apparatus) are uncorrected. IR spectra (Nujol mulls) were
Ild
12* C
WIN 51711
I
Y
0.81
0.13
>27.10
>271
0.08
0.54
2.18
>273
2.57
2.85
21.43
71
1.84
9.15
18.29
>284
3.51
31
o/H
2.35
1.76
recorded on a Perkin-Elmer 297 instrument. lH NMR spectra were recorded at 90 MHz on a Varian EM-390 spectrometer. Tetramethylsilane was used as an internal reference standard. All compounds were routinely checked by TLC and 'H NMR. NMR data were consistent with the indicated structures. TLC was performed with C. Erba silica gel Stratocrom SIF-254
Massa et al.
808 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 5
precoated plates. Developed plates were visualized by U V light. Merck silica gel 60 and alumina 90 were used for chromatographic purifications. Solvents were reagent grade and, when necessary, purified and dried by standard methods. Concentration of solutions after reactions and extractions involved the use of a rotary evaporator operating a t a reduced pressure of approximately 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. Microanalyses (within f 0 . 4 % of the theoretical values) were performed by the Microanalytical Laboratory of Prof. A. Pietrogrande, University of Padova, Italy. All compounds were analyzed for C, H, and N and, when present, C1 and S. Syntheses. Specific examples presented below illustrate general synthetic methods A-E. In general, samples prepared for physical (Table 1)and biological studies (Table 2-4) were dried in high vacuum over PzO5 for 20 h at temperatures ranging from 25 to 110 "C, depending on the sample melting point. Method A Example. 2-(5-Chloropentanoyl)-3-methylthiophene (5g) and 2-(5-chloropentanoyl)-4-methylthiophene (50. A solution of 5-chlorovaleryl chloride (15.5 g, 0.1 mol) and aluminum trichloride (13.3 g, 0.1 mol) in 1,2dichloroethane (150 mL) was added to a solution of 3-methylthiophene (40(9.8 g, 0.1 mol) in the same solvent (150 mL). The solution was stirred at room temperature for 2 h and poured onto a mixture of ice (200 g) and concentrated HCl(20 mL). Extractive workup with chloroform gave an oily residue which was chromatographed on a silica gel column eluting with 4% ethyl acetate in hexanes to yield 5g (9.0 9): 'H NMR (CC14)6 1.78-1.92 (m, 4H, CHZCHZCHZCHZ), 2.50 (s, 3H, CHd, 2.75-2.87 (m, 2H, COCH2), 3.48-3.58 (m, 2H, CH&l), 6.89 (d, J = 5.4 Hz, l H , thiophene H-4), 7.31 (d, J = 5.4 Hz, l H , thiophene H-5). Further elution of the above column afforded 5f (1.9 g): 'H NMR (CCl4) 6 1.77-1.88 (m, 4H, CHzCHzCH2CHz),2.23 (s, 3H, CH3), 2.75-2.88 (m, 2H, COCHz), 3.47-3.58 (m, 2H, CHZCl), 7.12 (d, J = 1.5 Hz, l H , thiophene H-3), 7.43 (d, J = 1.5 Hz, l H , thiophene H-5). Method B Example. 2-[5-[4-(4,5-Dihydro-2-oxazolyl)phenoxy]pentanoyl]-5-methylthiophene (7e). To a stirred solution of 5e (1.99 g, 9.2 mmol) in dry acetonitrile (100 mL) were added sodium iodide (1.12 g, 7.5 mmol), anhydrous potassium carbonate (1.42 g, 10.3 mmol), and 4-(4,5-dihydro2-oxazolyl)phenol(6a) hydrochloride (2.06 g, 10.3 mmol). The suspension was refluxed for 72 h and then filtered while hot, and the solution was evaporated. The residue was partitioned between ethyl acetate and water. The organic layer was washed successively with 5% aqueous NaOH, 5% aqueous sodium thiosulfate, water, and brine. Evaporation of the solvent left a solid residue, which was chromatographed on silica gel eluting with CHCl3:ethyl acetate (1:l) t o give pure 7e (0.79 g) as a white solid: lH NMR (CDC13) 6 1.83-1.95 (m, 4H, CHZCHZCHZCHZ), 2.50 (s, 3H, CH3), 2.83-2.97 (m, 2H, COCHz), 3.97-4.10 (superimposed signals, 4H, CHzN and CHzOAr), 4.30-4.40 (m, 2H, OCHzCHzN), 6.78 (d, l H , thiophene H-4), 6.87 (m, 2H, benzene H-2,6), 7.53 (d, l H , thiophene H-31, 7.88 (m, 2H, benzene H-3,5). 5-Chloro-2-(5-chloropentyl)thiophene (10d). A solution of 5d (1.19 g, 5 mmol) and aluminum trichloride (0.66 g, 5 mmol) in a mixture of anhydrous diethyl ether (40 mL) and anhydrous THF (30 mL) was slowly added to a cooled (5 "C) suspension of lithium aluminum hydride (0.38 g, 10 mmol) and aluminum trichloride (1.33 g, 10 mmol) in diethyl ether (10 mL). The mixture was stirred a t 30-40 "C for 2 h, and then the reaction was cautiously quenched with water (20 mL) and 6 N HzS04 (20 mL). The organic layer was separated, washed twice with brine, dried, and evaporated to furnish 10d (0.99 g) as an oil homogeneous by TLC analysis (SiOdbenzene). Method C Example. 2-[5-(4-Carboxyphenoxy)pentanoyll-5-methylthiophene (13e). A mixture of 8e (1.6 g, 4.6 mmol), 1.5 N NaOH (15.3 mL, 23 mmol), and EtOH (15 mL) was heated a t 90 "C for 2.5 h with stirring. After cooling, the solution was diluted with water (100 mL) and made acidic by adding 2 N HC1. Extraction with ethyl acetate followed by usual workup gave a solid, which was recrystallized to afford pure 13e (1.4 g).
3-(5-Chloropentanoyl)-lH-pyrrole (16). A solution of 15 (6.52 g, 0.02 mol) in dioxane (75 mL) and 5 N NaOH (70 mL, 0.35 mol) was stirred at room temperature for 17 h. The organic layer was separated and the aqueous one extracted with ethyl acetate. The combined organic solution was washed with brine, dried, and evaporated to leave a brown solid, which was recrystallized t o give 16 (3.16 g). Method D Example. 3-[5-[4-(4,5-Dihydro-2-oxazolyl)phenoxy]pentanoyl] 1,2,54rimethyl-llY-pyrrole (22d).A solution of diethyl azodicarboxylate (DEAD) (1.25 g, 7.2 mmol) in anhydrous THF (20 mL) was added over a period of 10 min t o a solution of 21b (1.51 g, 7.2 mmol), 6a (1.17 g, 7.2 mmol), and triphenylphosphine (1.89 g, 7.2 mmol) in anhydrous THF (100 mL). After stirring a t room temperature for 18 h, the solvent was removed under reduced pressure, and the residue was partitioned between water and CHC13. The organic layer was washed twice with 2 N KOH and then with brine, dried, and evaporated. Column chromatography on silica gel (ethyl acetate as eluent) provided pure 22d (2.35 g). 4- (5-Chloropentanoyl)- 1-methyl-lH-pyrrole-2-carboxaldehyde (23). To a cooled (0-5 "C) solution of DMF (0.78 mL, 10 mmol) in 1,2-dichloroethane (20 mL) was added over a period of 5-10 min a solution of oxalyl chloride (1.27 g, 10 mmol) in 1,2-dichloroethane (20 mL). After stirring a t room temperature for 15 min, the suspension was cooled (0-5 "C) again and treated with a solution of 1-methyl-1H-pyrrole (4b) (0.81 g, 10 mmol) in 1,2-dichloroethane (20 mL). The mixture was stirred a t room temperature for 15 min and then treated with aluminum trichloride (2.92 g, 22 mmol) and 5-chlorovaleryl chloride (1.55 g, 10 mmol). After 3 h, the reaction mixture was poured onto crushed ice (100 g) containing 50% NaOH (10 mL) and stirred for 10 min. The pH of the solution was adjusted t o 4 with concentrated HCl, the organic layer was separated, and the aqueous one was extracted with CHCl3. The combined organic solution was washed with water, dried, and evaporated t o dryness. The brown oily residue was purified by chromatography on silica gel (CHC13) t o afford 23 (1.81 g). Method E Example. 1-[5-[4-(4,5-Dihydro-2-oxazolyl)phenoxylpentyll-lH-pyrrole(28). A solution of lH-pyrrole (2.01 g, 30 mmol) in anhydrous dioxane (20 mL) was added dropwise to a suspension of 97% NaH (0.82 g, 33 mmol) in 20 mL of the same solvent. The mixture was refluxed for 15 min and then cooled again to room temperature. A solution of 27 (2.34 g, 7.5 mmol) was added dropwise t o the above mixture, which was then refluxed for 5 h. After cooling, the reaction was quenched with a saturated solution of NH&1(50 mL) and the mixture concentrated and extracted with ethyl acetate. The organic solution was dried and evaporated to give a residue, which was chromatographed on alumina eluting with CHC13 and recrystallized to afford pure 28 (1.19 g). Antiviral Assays. HeLa-Ohio cells were grown a t 37 "C in a 5% COz atmosphere in Dulbecco's modified medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 UI/ mL penicillin G, and 100 pg/mL streptomycin. Cultures were checked periodically for the absence of mycoplasma contamination with a MicoTect kit (Gibco). Compounds were solubilized in DMSO as 200x stock solutions and then serially diluted in maintenance medium t o achieve the desired final concentrations. Anti-HRV assays were based on the inhibition of virusinduced cytopathogenicity. Briefly, 20 000 HeLa cells were seeded in each well of flat-bottomed microtiter trays in 50 mgl mL D-MEM containing 2% fetal calf serum, MgClz (30 mM), and DEAE-dextran (15 pg/mL). The cells were then allowed to form a subconfluent monolayer by incubating overnight a t 37 "C in a humidified incubator under a n atmosphere of 5% COz. Maintenence medium (50 mL) containing various concentrations of the test compounds was then added followed by 20 mL of an H'RV suspension calibrated to produce complete cytopathic effect within 72 h after infection [ZOO-2000 infectious virus particles (PFU)/monolayer]. Following incubation a t 33 "C, viability of the HeLa cells was determined by the 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M'M') method.
-
Thienyl and Pyrryl Compounds with Antirhinovirus Activity
Journal of Medicinal Chemistry, 1995, Vol. 38, No. 5 809
Cytotoxicity of test compounds was evaluated in parallel with their antiviral activity. It was based on the viability of mock-infected cells, as monitored by the MTT method after a 72 h incubation at 37 “C.
(7) Diana, G. D.; Cutcliffe, D.; Oglesby, R. C.; Otto, M. J.; Mallamo, J. P.; Akullian, V.; McKinlay, M. A. Synthesis and StructureActivity Studies of Some Disubstituted Phenylisoxazoles against Human Picornavirus. J. Med. Chem. 1989,32,450-455. (8) Diana, G. D.; Treasurywala, A. M.; Bailey, T. R.; Oglesby, R. C.; Pevear, D. C.; Dutko, F. J. A Model for Compounds Active against Human Rhinovirus-14 Based on X-ray Crystallography Data. J. Med. Chem. 1990,33,1306-1311. (9) Fox, M. P.; Otto, M. J.; McKinlay, M. A. Prevention of Rhinovirus and Poliovirus Uncoating by WIN 51711, a New Antiviral Drug. Antimicrob. Agents Chemother. 1986,30, 110-116. (10)Pevear, D. C.; Fancher, M. J.; Felock, P. J.; Rossmann, M. G.; Miller, M. S.; Diana, G. D.; Treasurywala, A. M.; McKinlay, M. A,; Dutko, F. J. Conformational Change in the Floor of the Human Rhinovirus Canyon Blocks Adsorption to HeLa Cell Receptors. J. Virol. 1989, 63, 2002-2007. (11) Smith, T. J.; Kremer, M. J.; Luo, M.; Vriend, G.; Arnold, E.; Kamer, G.; Rossmann, M. G.; McKinlay, M. A.; Diana, G. D.; Otto, M. J. The Site of Attachment in Human Rhinovirus-14 for Antiviral Agents That Inhibit Uncoating. Science 1986, 233, 1286-1293. (12) Massa, S.; Artico, M.; Mai, A.; Ragno, R.; Corelli, F.; Pani, A.; Marongiu, M. E.; Tramontano, E.; La Colla, P. Synthesis of New Disoxaril Analogues with Potent and Selective Anti-Human Rhinovirus-14 Activity. BioMed. Chem. Lett. 1991,1,575-578. (13) Xu, R. X.; Anderson, H. J.; Gogan, N. J.;Loader, C. E.; McDonald, R. Pyrrole Chemistry XXV: A Simplified Synthesis of Some 3-Substituted Pyrroles. Tetrahedron Lett. 1981,22,4899-4900. (14) Kakushima, M.; Hamel, P.; Frenette, R.; Rokach, J. Regioselective Synthesis of Acylpyrroles. J. Org. Chem. 1983,48, 32143219. (15) Anderson, H. J.; Loader, C. E. The Synthesis of 3-Substituted Pyrroles from Pyrrole. Synthesis 1985, 353-364. (16) Massa, S.; Artico, M.; Corelli, F.; Mai, A,; Di Santo, R.; Cortes, S.; Marongiu, M. E.; Pani, A,; La Colla, P. Synthesis and Antimicrobial and Cytotoxic Activities of Pyrrole-Containing Analoguesof Trichostatin A. J.Med. Chem. 1990,33,2845-2849.
Acknowledgment. This work has been supported by Cenci Bolognetti-Institute Pasteur Foundation (in part) and Italian MURST (60% fund, Dpt Studi Farmaceutici), Italian CNR (Dpt Farmaco Chimico Tecnologico), and Regione Autonoma della Sardegna (Dpt Biologia Sperimentale, Sezione Microbiologia).
References (1)Presented in part as a poster communication at the Fourth
International Conference on Antiviral Research, New Orleans,
LA, April 21-26, 1991.
(2) Hamparian, V. V.; Colonno, R. J.; Cooney, M. K.; Dick, E. K; Gwaltnev. J. M.: Huehes. J. H.: Jordan. W. S.: Kanikian. A. Z.: Mogabgib, W. J.; Minto; A.; Phillips, ’C. A.;’Re&kert,’R. R.; Schieble, J. H.; Stott, E. J.; Tyrrell, D. A. J. A Collaborative Report: Rhinoviruses-Extension of the Numbering System from 89 to 100. Virology 1987, 159, 191-192. (3) Diana, G. D.; McKinlay, M. A.; Brisson, C. J.; Zalay, E. S.; Miralles, J. V.; Salvador, U. J. Isoxazoles with Antipicornavirus Activity. J. Med. Chem. 1988,28, 748-752. (4) Diana, G. D.; McKinlay, M. A.; Otto, M. J.;Akullian, V.; Oglesby, C. [[(4,5-Dihydro-2-oxazolyl)phenoxylalkyllisoxazoles.Inhibitors of Picornavirus Uncoating. J.Med. Chem. 1986,28,1906-1910. (5) Diana, G. D.; Oglesby, R. C.; Akullian, V.; Carabateas, P. M.; Cutcliffe, D.; Mallamo, J. P.; Otto, M. J.; McKinlay, M. A.; Maliski, E. G.; Michalec, S. J. Structure-Activity Studies of 544(4,5-Dihydro-2-oxazolyl)phenoxy]alkyl]-3-methylisoxazoles: Inhibitors of Picornavirus Uncoating. J.Med. Chem. 1987,30, 383-388. (6) Diana, G. D.; Otto, M. J.; Treasurywala, A. M.; McKinlay, M. A.; Oglesby, R. C.; Maliski, E. G.; Rossmann, M. G.; Smith, T. J. Enantiomeric Effects of Homologues of Disoxaril on the Inhibitory Activity against Human Rhinovirus-14. J. Med. Chem. 1988,31,540-544.
Jhf930828E
